Metabolite profiling of the effects of diabetes in rat retina by 1H NMR spectroscopy

Abstract

Abstract: : Purpose: It has been shown that the metabolism of glutamate and GABA are altered in diabetic retinas, indicating that diabetes induces metabolic changes in retinas. In the present work, we monitored changes in the levels of intermediary metabolites in diabetic rat retinas, with a powerful technique (1H NMR spectroscopy), to check which metabolic pathways could be mainly affected by diabetes in retina. Methods: Diabetes was induced in 2–month old male Wistar rats with a single i.p. injection of streptozotocin (STZ, 65 mg/kg). Retinal extracts were obtained from 1 week, 1 month and 3 months diabetic rats, and from age–matched controls. 1H NMR spectra of retinal extracts were recorded on an 11.7 T Varian Unity spectrometer, operating at 499.8 MHz, using a 5 mm switchable NMR probe. Metabolite identification was made by comparison of the 1H NMR spectra of retinal extracts with the individual spectra of metabolites and by comparison with published tables of 1H chemical shifts. Metabolite levels were evaluated by spectral deconvolution of each resonance from the 1H NMR spectra and comparison with the intensity of the formic acid resonance, used as an internal standard. Results: Retinas isolated from STZ–treated rats showed no significant differences in the levels of several intermediary metabolites (e.g., glutamate, glutamine, GABA, alanine, pyruvate) when compared to control animals, in the periods tested. As expected, significant increases in the levels of glucose were observed for the various periods studied. It was observed a tremendous increase (∼250 times) in the levels of acetoacetate in retinas from 1 month diabetic rats, as compared to controls of the same age. Other metabolites, such as lactate, showed a consistent decrease in their levels in diabetic retinas, while others like acetate showed a consistent increase, but no significance was attained for any of the time periods studied. Conclusions: These results show that diabetes does not induce significant changes in the majority of intermediary metabolites in rat retinas, as assessed by 1H NMR spectroscopy, although some metabolites may be affected.

Keywords: diabetic retinopathy • metabolism • retina